Protein Kinase A-Dependent Phosphorylation Modulates β₁Pix Guanine Nucleotide Exchange Factor Activity through 14-3-3β Binding

Abstract

β₁Pix is a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42 which has been shown to mediate signaling pathways leading to cytoskeletal reorganization. In the present study, we show that the basal association between endogenous βPix and endogenous 14-3-3β was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3β and a β₁Pix mutant that is insensitive to protein kinase A phosphorylation, β₁Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3β to β₁Pix results in inhibition of Rac1 GTP loading in 293 cells and in vitro. Furthermore, we show that deletion of 10 amino acid residues within the leucine zipper domain is sufficient to block β₁Pix homodimerization and 14-3-3β binding and modulates β₁Pix-GEF activity. These residues also play a crucial role in β₁Pix intracellular localization. These results indicate that 14-3-3β negatively affects the GEF activity of dimeric β₁Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3β in modulating β₁Pix-GEF activity.

ACKNOWLEDGMENTS

This work was supported in part by an American Heart Association grant (0520114Z) to A.C. and by National Institutes of Health grant HL22563 to A.S.